Received: 22 December 2017; Accepted: 1 February 2018; Published: 3 February 2018

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1 viruses Article A Simple Mechanism Based on Amino Acid Substitutions is not a Critical Determinant of High Mortality of Japanese Encephalitis Virus Infection in Mice Yuki Takamatsu 1,, Leo Uchida 1,, Muhareva Raekiansyah 1, Mark Anthony Luz 1, Kouichi Morita 1,2 and Daisuke Hayasaka 1,2,3, * 1 Department of Virology, Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki , Japan; yuki.takamatsu@staff.uni-marburg.de (Y.T.); uchidaleo@rakuno.ac.jp (L.U.); m.raekiansyah@gmail.com (M.R.); markanthonyluz@gmail.com (M.A.L.); moritak@nagasaki-u.ac.jp (K.M.) 2 Leading Graduate School Program, Nagasaki University, Nagasaki , Japan 3 Center for Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki , Japan * Correspondence: hayasaka@nagasaki-u.ac.jp; Tel.: ; Fax: Current Affiliation: Institute of Virology, Philipps University Marburg, Marburg, Germany. Current Affiliation: Laboratory of Zoonotic Diseases, Division of Health and Environmental Science, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu , Japan. Received: 22 December 2017; Accepted: 1 February 2018; Published: 3 February 2018 Abstract: For the development of effective treatment strategies for Japanese encephalitis (JE), it is important to identify the viral factors causing severe disease during JE virus (JEV) infection. In this study, we assessed whether amino acid substitutions are critical factors for higher mortality of JaTH160 compared with JaOArS982 in mice using the technique of infectious cdna clones. We raised the possibility that two amino acids of C 124 and NS3 482 of JaTH160 may contribute to increased mortality in mice. However, simultaneous substitutions of these amino acids did not significantly increase the virulence of JaOArS982, suggesting that high mortality due to JaTH160 viral infection cannot be simply attributed to the specific amino acids. Multiple and complex, but not simple, mechanisms may induce the high mortality of JaTH160 infection in mice. Keywords: Japanese encephalitis virus; pathogenicity; mouse; recombinant; amino acid substitution 1. Introduction Japanese encephalitis (JE) virus (JEV) causes approximately 30,000 to 50,000 cases and 10,000 to 15,000 deaths in Asian countries annually [1,2]. JEV belongs to the family Flaviviridae, genus Flavivirus [3,4], whose genomic RNA encodes three structural (C, prm, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins [5]. The clinical symptoms of JE vary from mild to severe disease, including a non-specific febrile illness, meningitis, encephalitis and meningoencephalitis [6,7]. However, the mechanism of severe central nervous system (CNS) disease has not been fully elucidated. To evaluate the disease pathogenesis in JEV infection, mice have been employed as a useful infection model [8 10]. Mouse models of JEV infection have suggested that several viral and host factors affect disease severity during JEV infection. We previously showed that subcutaneous infection with the JaTH160 strain of JEV causes significantly higher mortality and more pronounced virus propagation in the brains of mice compared with those of the JaOArS982 strain [9]. Thus, we have considered that genome-based comparisons between JaTH160 and JaOArS982 strains would provide Viruses 2018, 10, 62; doi: /v

2 Viruses 2018, 10, 62 2 of 10 useful information for identifying the viral factors responsible for the pathogenicity in JEV infection in vivo. Previous studies have demonstrated that amino acid substitutions are major factors affecting the virulence in flavivirus infections. For example, it has been shown that amino acid substitutions in the E protein, such as E 49, E 123, E 138, E 176, E 306, and E 389/390 affect the virulence of flaviviruses in the CNS [11 14], and amino acids, such as C 42,43, prm 15,17, NS2A 23, and NS ,891, contribute to the flavivirus pathogenicity in mice [15 18]. We have also focused on the amino acid substitutions between JaTH160 and JaOArS982 strains to elucidate the pathogenicity in our previous studies. JaTH160, but not JaOArS982, expresses the NS1 protein and that NS1 enhances JEV production in avian cells and embryonated chicken eggs [19]. Previous studies have shown that the NS1 protein plays a role in the enhanced virulence of the JEV SA14 strain in mice [20]. Thus, we have predicted that NS1 protein is a critical factor of the higher pathogenicity of JaTH160 compared with JaOArS982. However, our data suggested that NS1 protein expression in the JaOArS982 strain reduces the mortality in mice, suggesting that the effect of NS1 on pathogenicity in vivo may vary among virus strains [21]. We also identified a unique amino acid of NS2A 113 phenylalanine that affects the efficient propagation of JaTH160 in neuroblastoma Neuro-2a cells, but not extraneural origin cells compared with the JaOArS982 strain [22]. Therefore, we predicted that this NS2A 113 phenylalanine is responsible for the high pathogenicity of JaTH160. However, this amino acid did not affect viral loads in the brain nor survival curves in mice, suggesting that virus propagation in vitro may not reflect the level of virus neurovirulence in vivo [22]. There are 19 amino acid differences between JaTH160 and JaOArS982. Therefore, in the present study, we aimed to identify the amino acids affecting the virulence and to assess whether those amino acid substitutions are critical to determine the high mortality due to JaTH160 infection by a reductionist approach using the infectious cdna clone techniques of JaTH160 and JaOArS982 strains. 2. Materials and Methods 2.1. Ethical Statement The animal experiment protocols were approved by the Animal Care and Use Committee of Nagasaki University (approval number: / December , / August ). The animal experiments were performed in accordance with the recommendations in the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology Viruses and Cells The full-length cdna clones S982-IC and JaTH-IC were developed from the genome sequences of JaOArS982 and JaTH160 strains, respectively, as described previously [19]. Based on the S982-IC and JaTH-IC, recombinant JEV clones were constructed at the unique restriction enzyme sites, and the point mutations were introduced by site-directed mutagenesis as described previously [19]. The plasmids containing the full-length cdna clones were linearized and transcribed into RNA using an SP6 transcription kit (mmessage mmachine SP6 kit, Life Technologies, Carlsbad, CA, USA) [23]. The RNAs were introduced into baby hamster kidney (BHK) cells by electroporation. Infectious viruses were recovered from those cell culture fluids. Virus stocks were stored at 80 C until they were used. Viral titers were determined by plaque-forming assays using BHK cells and were expressed as plaque forming unit (PFU)/mL [24]. BHK cells were maintained in Eagle s Minimal Essential Medium (EMEM) containing 10% fetal calf serum (FCS) and antibiotics.

3 Viruses 2018, 10, 62 3 of Mice C57BL/6j (B6) mice were purchased from Japan SLC, Inc, or Japan CLEA, Inc. Five- to seven-week-old mice were subcutaneously inoculated with 10 4 PFU of JEV diluted in EMEM containing 2% FCS. Mock infected mice were inoculated with supernatant of cultured BHK cells. Mice were weighed daily, and survivals were recorded for 21 days. Viruses 2018, 10, x FOR PEER REVIEW 3 of Statistical Analyses FCS. Mock infected mice were inoculated with supernatant of cultured BHK cells. Mice were weighed The log-rank daily, and (Mantel-Cox) survivals were recorded test wasfor used 21 days. for statistical analysis to assess the significant differences of survival curves between each recombinant virus and S982-infectious clone (IC) virus. A p value of 2.4. Statistical Analyses <0.05 was considered statistically significant. The log-rank (Mantel-Cox) test was used for statistical analysis to assess the significant differences of survival curves between each recombinant virus and S982-infectious clone (IC) virus. 3. Results A p value of <0.05 was considered statistically significant Separate 3. Results Regions of Viral Genome Affect the High Mortality in Mice Amino 3.1. acid Separate differences Regions of Viral between Genome Affect JaOArS982 the High Mortality and JaTH160 in Mice are shown in Figure 1A. S982-IC and JaTH-IC viruses showed 47 and 100% mortality in mice, respectively (Figure 1B). To address Amino acid differences between JaOArS982 and JaTH160 are shown in Figure 1A. S982-IC and the aminojath-ic acids affecting viruses showed the 47 different and 100% levels mortality of virulence in mice, respectively between(figure these1b). viruses, To address wethe applied the reductionist amino approach, acids affecting whichthe introduces different levels JaTH-IC of virulence viral genome between these regions viruses, intowe S982-IC, applied and the assessed those recombinant reductionist viruses approach, bywhich whether introduces mortality JaTH-IC reached viral genome highregions rates, into ideally S982-IC, 100%. and We assessed expected this those recombinant viruses by whether mortality reached high rates, ideally 100%. We expected this approach to narrow down the regions of viral genome stepwise and finally determine the amino acids approach to narrow down the regions of viral genome stepwise and finally determine the amino of JaTH160acids responsible of JaTH160 for responsible the high for mortality the high mortality in mice. in mice. Figure 1. Mortalities of mice infected with recombinant viruses of JaOArS982 and JaTH160 replacing four distinct regions of the viral genome. (A) schematic representation of 19 amino acid differences of

4 Viruses 2018, 10, 62 4 of 10 Viruses 2018, 10, x FOR PEER REVIEW 4 of 10 recombinant S982-IC and JaTH-IC viruses derived from JaOArS982 and JaTH160, respectively. Numbers Figure 1. Mortalities of mice infected with recombinant viruses of JaOArS982 and JaTH160 replacing indicate the position of amino acid in each responsible protein, respectively. (B) Survival curves and four distinct regions of the viral genome. mortality rates of mice infected with 10 4 (A) schematic representation of 19 amino acid differences of recombinant S982-IC and JaTH-IC viruses pfuderived of S982-IC from (n JaOArS982 = 15) and and JaTH-IC JaTH160, (n = respectively. 15) for 21 days. Opened Numbers and closed indicate diamonds the position indicate of amino S982-IC acid in and each JaTH-IC, responsible respectively. protein, respectively. The log-rank (B) Survival (Mantel-Cox) test was curves usedand for mortality statistical rates analysis of mice infected to assess with significant 10 4 pfu of differences S982-IC (n = 15) of and survival JaTH-IC curves. (n = 15) Afor p value 21 of <0.05 was days. considered Opened and statistically closed diamonds significant. indicate S982-IC (C) Survival and JaTH-IC, curvesrespectively. and mortality The log-rank rates of (Mantel- mice infected with 10 Cox) 4 pfu test of was S-J1 used (n for = 10), statistical S-J2 (n analysis = 10), to S-J3 assess (n significant = 9) and differences S-J4 (n = 9) of viruses survival curves. based on A p S982-IC, value in of <0.05 was considered statistically significant. (C) Survival curves and mortality rates of mice which genome regions 1 (5 untranslated region (UTR)-NS1 323 ), 2 (NS NS3 35 ), 3 (NS3 36 -NS5 567 ), infected with 10 and 4 (NS UTR) 4 pfu of S-J1 (n = 10), S-J2 (n = 10), S-J3 (n = 9) and S-J4 (n = 9) viruses based on S982- were replaced with those of S982-IC, respectively. Closed triangles indicate IC, in which genome regions 1 (5 untranslated region (UTR)-NS1323), 2 (NS1324-NS335), 3 (NS336- recombinant viruses. The black inverted triangles indicate the positions and numbers of amino acids NS5567), and 4 (NS UTR) were replaced with those of S982-IC, respectively. Closed triangles derived indicate from JaTH-IC. recombinant viruses. The black inverted triangles indicate the positions and numbers of amino acids derived from JaTH-IC. We first constructed four recombinant viruses (S-J1, S-J2, S-J3 and S-J4) based on S982-IC by introducing We four first regions constructed of JaTH-IC four recombinant (Figure 1C). viruses Mortalities (S-J1, S-J2, of S-J1-, S-J3 and S-J2-, S-J4) S-J3-, based andon S-J4-infected S982-IC by mice were 80, introducing 40, 50, and four 67%, regions respectively, of JaTH-IC and (Figure S-J11C). virus Mortalities infection of was S-J1-, found S-J2-, S-J3-, to cause and S-J4-infected higher mortality mice were 80, 40, 50, and 67%, respectively, and S-J1 virus infection was found to cause higher than others (Figure 1C). mortality than others (Figure 1C). We next constructed recombinant viruses of J-S1, J-S2, J-S3, and J-S4 based on JaTH-IC by replacing We next constructed recombinant viruses of J-S1, J-S2, J-S3, and J-S4 based on JaTH-IC by the four replacing regions the of four S982-IC regions (Figure of S982-IC 2A). (Figure Infections 2A). Infections with J-S2with andj-s2 J-S4 and viruses J-S4 viruses caused caused and 91% mortality and 91% in mice, mortality respectively in mice, respectively (Figure 2A). (Figure On the 2A). other On the hand, other infections hand, infections with with J-S1 J-S1 and and J-S3 J-S3 viruses resulted viruses in 55resulted and 73% in 55 mortality and 73% in mortality mice, respectively in mice, respectively (Figure (Figure 2A). 2A). Figure 2. Mortalities of mice infected with recombinant viruses based on S982-IC and JaTH-IC. (A) Survival curves and mortality rates of mice infected with 10 4 pfu of J-S1 (n = 11), J-S2 (n = 13), J-S3

5 Viruses 2018, 10, 62 5 of 10 (n = Viruses 11), 2018, and10, J-S4 x FOR (n PEER = 11) REVIEW viruses based on JaTH-IC, in which genome regions 1, 2, 3, and5 4of were 10 replaced with those of S982-IC, respectively. (B) Survival curves and mortality rates of mice infected with 10 4 Figure 2. Mortalities of mice infected with recombinant viruses based on S982-IC and JaTH-IC. (A) pfu of S-J13 (n = 10) and S-J24 (n = 10), whose genome regions 1 and 3, or regions 2 and 4, Survival curves and mortality rates of mice infected with 10 4 pfu of J-S1 (n = 11), J-S2 (n = 13), J-S3 (n were of JaTH-IC. Closed triangles indicate recombinant viruses. The black inverted triangles indicate = 11), and J-S4 (n = 11) viruses based on JaTH-IC, in which genome regions 1, 2, 3, and 4 were replaced the positions with those andof numbers S982-IC, respectively. of amino acids (B) Survival derivedcurves fromand JaTH-IC. mortality rates of mice infected with 10 4 pfu of S-J13 (n = 10) and S-J24 (n = 10), whose genome regions 1 and 3, or regions 2 and 4, were of From these JaTH-IC. results, Closed we triangles postulated indicate recombinant that a combination viruses. The of black distinct inverted regions triangles of indicate 5 UTR-NS1 the 323 and NS3 36 -NS5 positions 567 mayand contribute numbers of amino to theacids high derived mortality from JaTH-IC. due to JaTH-IC virus infection. Thus, we examined the mortality due to S-J13 virus infection in mice, whose regions of 5 UTR-NS1 323 and From these results, we postulated that a combination of distinct regions of 5 UTR-NS1323 and NS3 36 -NS5 NS336-NS weremay of JaTH-IC, contribute and to the other high mortality regionsdue were to JaTH-IC of S982-IC virus (Figure infection. 2B). Thus, The we mortality examined rate of S-J13 virus-infected the mortality due mice to S-J13 was 80%, virus infection although in the mice, survival whose regions curveof was 5 UTR-NS1323 not significantly and NS336-NS5567 different from that of S982-IC were of JaTH-IC, virus (Figure and other 2B). regions were of S982-IC (Figure 2B). The mortality rate of S-J13 virusinfected mice it iswas controversial 80%, although asthe tosurvival whether curve thewas virulence not significantly of S-J13 different virus from is significantly that of S982- higher Although IC virus (Figure 2B). than that of S982-IC virus, we further attempted to focus narrowly on the site of 5 UTR-NS1 323 and Although it is controversial as to whether the virulence of S-J13 virus is significantly higher than NS3 36 -NS5 that 567 of S982-IC. Thus, virus, we further we further constructed attempted recombinant to focus narrowly viruses on the by site dividing of the 5 UTR-NS1 323 and 5 UTR-NS1323 and NS336- NS3 36 -NS5 NS Thus, regions we into further three constructed and tworecombinant regions, respectively. viruses by dividing the 5 UTR-NS1323 and NS336- Recombinant NS5567 regions viruses into three ofand S-J13a two regions, and S-J13b respectively. were constructed as containing NS3 36 -NS4B 110 and NS4B 111 -NS5 Recombinant 567 of JaTH-IC viruses based of S-J13a on S-J1, and respectively S-J13b were constructed (Figure 3A). as containing NS336-NS4B110 Mortality of S-J13a virus-infected and NS4B111-NS5567 of JaTH-IC based on S-J1, respectively (Figure 3A). Mortality of S-J13a virus-infected mice was 100%, whereas that of S-J13b infected mice was 20% (Figure 3A). On the other hand, following mice was 100%, whereas that of S-J13b infected mice was 20% (Figure 3A). On the other hand, infections with recombinant viruses of S-J1a3, S-J1b3, and S-J1c3 containing 5 UTR-M, M 153 -E 303, following infections with recombinant viruses of S-J1a3, S-J1b3, and S-J1c3 containing 5 UTR-M152, or E 304 -NS1 M153-E303, 323 of or JaTH-IC based on S-J3, respectively, mortalities were 80, 70, and 60%, respectively, E304-NS1323 of JaTH-IC based on S-J3, respectively, mortalities were 80, 70, and 60%, and S-J1a3 respectively, infectionand showed S-J1a3 infection a highershowed mortality a higher (Figure mortality 3B). (Figure 3B). Figure 3. Mortalities of mice infected with S-J13-based recombinant viruses. (A) Survival curves and mortality rates of mice infected with 10 4 pfu of S-J13a (n = 10) and S-J13b (n = 5) and 3b (NS4B 111 -NS5 567 )

6 Viruses 2018, 10, 62 6 of 10 Viruses 2018, 10, x FOR PEER REVIEW 6 of 10 Figure 3. Mortalities of mice infected with S-J13-based recombinant viruses. (A) Survival curves and mortality rates of mice infected with 10 were replaced with those of JaTH-IC. (B) 4 pfu of S-J13a (n = 10) and S-J13b (n = 5) based on S-J13, in Survival curves and mortality rates of mice infected with 10 4 which regions 3a (NS336-NS4B110) and 3b (NS4B111-NS5567) were replaced with those of JaTH-IC. (B) pfu of S-J1a3 (n = 10), S-J1b3 (n = 10), and S-J1c3 (n = 5) based on S-J13, in which regions 1a (5 UTR-M 152 ), Survival curves and mortality rates of mice infected with 10 1b (M 153 -E 303 ), and 1c (E 304 -NS1 323 ) were replaced with those 4 pfu of S-J1a3 (n = 10), S-J1b3 (n = 10), and of JaTH-IC. Closed triangles indicate S-J1c3 (n = 5) based on S-J13, in which regions 1a (5 UTR-M152), 1b (M153-E303), and 1c (E304-NS1323) were recombinant viruses. The black inverted triangles indicate the positions and numbers of amino acids replaced with those of JaTH-IC. Closed triangles indicate recombinant viruses. The black inverted derived from JaTH-IC. triangles indicate the positions and numbers of amino acids derived from JaTH-IC. From From these these results, results, we we further further focused focused on on the the amino amino acids acids in in the the 5 UTR-M 152 and NS3 36 -NS4B 5 UTR-M152 and NS336-NS4B regions, regions, where where viral viral genome genome sequences of of JaTH160 may induce high highmortality mortalityin inmice. mice Amino Amino Acids Acids C 124 and NS3 482 of JaTH160 May Affect the High Mortality in Mice C124 and NS3482 of JaTH160 May Affect the High Mortality in Mice To To focus focus on on amino amino acid acid substitutions, we next constructed recombinant recombinant viruses viruses based based on on S-J3 S-J3 and and S-J1 S-J1 by by inserting a point mutation mutation that that substituted substituted the amino the amino acids in acids each in5 UTR-M152 each 5 UTR-M and NS and NS3 NS4B NS4B region. 110 region. There There are are two two amino acid differences in the 5 UTR-M region between S982-IC and and JaTH-IC. JaTH-IC. We We constructed S-JC S-JC1243a and S-JM1403a as containing a point mutation of the position C124 C 124 and andm140 M 140 based on on S-J3, respectively (Figure 4A). After infections with these viruses, mortalities of of mice mice were were and and 80%, 80%, respectively (Figure 4A). Figure 4. Cont.

7 Viruses 2018, 10, 62 7 of 10 Viruses 2018, 10, x FOR PEER REVIEW 7 of 10 Figure 4. Mortalities Figure 4. of Mortalities mice infected of mice withinfected recombinant with viruses recombinant inserting viruses pointinserting mutations. point (A) mutations. Survival (A) curves andsurvival mortality curves ratesand of mice mortality infected rates with of mice 10 4 infected pfu of S-JC with a pfu (n of = 10) S-JC1243a and S-JM (n = 10) 140 3a and (n S-JM1403a = 10) (n viruses based = 10) onviruses S982-IC based containing S982-IC genome containing region genome 3a of JaTH-IC, region 3a in of which JaTH-IC, amino which acidsamino C acids C and and M140 M were replaced with those of JaTH-IC, respectively. (B) Survival curves and mortality rates 140 were replaced with those of JaTH-IC, respectively. (B) Survival curves and mortality rates of of mice infected with 10 4 pfu of S-J1aNS3323 (n = 5), S-J1aNS3337 (n = 8), S-J1aNS3482 (n = 10), S-J1aNS3562 mice infected with 10 4 pfu of S-J1aNS3 323 (n = 5), S-J1aNS3 337 (n = 8), S-J1aNS3 482 (n = 10), S-J1aNS3 562 (n = 5), S-J1aNS4A3 (n = 10), and S-J1aNS4B18 (n = 5), S-J1aNS4A (n = 5) viruses based on S982-IC containing genome 3 (n = 10), and S-J1aNS4B 18 (n = 5) viruses based on S982-IC containing genome region 1a of JaTH-IC, in which amino acids NS3323, NS3337, NS3482, NS3562, NS4A3, and NS4B18 region 1a of JaTH-IC, in which amino acids NS3 were 323, NS3 337, NS3 482, NS3 562, NS4A 3, and NS4B 18 were replaced with those of JaTH-IC, respectively. (C) Survival curves and mortality rates of mice infected replaced with those of JaTH-IC, respectively. (C) Survival curves and mortality rates of mice infected with 10 4 pfu of S-J C124NS3482 (n = 10) virus based on S982-IC, whose amino acid positions of C124 and with 10 4 pfu of S-J C 124 NS3 482 (n = 10) virus based on S982-IC, whose amino acid positions of C 124 NS3482 were of JaTH160-IC. Closed triangles indicate recombinant viruses. The black inverted and NS3 482 were of JaTH160-IC. Closed triangles indicate recombinant viruses. The black inverted triangles indicate the positions and numbers of amino acids derived from JaTH-IC. triangles indicate the positions and numbers of amino acids derived from JaTH-IC. On the other hand, there are six amino acid differences in the NS336-NS4B110 region between On the S982-IC other hand, JaTH-IC there are (Figure six amino 1A). Thus, acid differences we constructed in therecombinant NS3 36 -NS4Bviruses 110 region of S-J1aNS3323, between S- S982-IC and J1aNS3337, JaTH-IC S-J1aNS3482, (Figure S-J1aNS3562, 1A). Thus, S-J1aNS4A3, we constructed and S-J1aNS4B18, recombinant to contain viruseseach of S-J1aNS3 point mutation 323, of S-J1aNS3 337 the, S-J1aNS3 position NS3323, 482, S-J1aNS3 NS3337, 562 NS3482,, S-J1aNS4A NS3562, NS4A3, 3, and S-J1aNS4B and NS4B18 18 of, to JaTH160 containbased each on point S-J1a, mutation respectively of the position (Figure NS3 4B). 323 Following, NS3 337, infections NS3 482, NS3 with 562 these, NS4A viruses, 3, and mortalities NS4B 18 of ofinfected JaTH160 mice based were on 20, S-J1a, 63, 90, 60, respectively 70, (Figure and 40%, 4B). respectively Following(Figure infections 4B). with these viruses, mortalities of infected mice were 20, 63, 90, 60, 70, and These 40%, results respectively suggested (Figure that the 4B). amino acids C124 and NS3482 of JaTH-IC may contribute to the Thesehigh results mortality suggested of JaTH-IC that the infection, amino and acids provide C 124 and the NS3 possibility 482 of JaTH-IC that a combination may contribute of amino to the acids of C124 and NS3482 high mortality of JaTH-ICof infection, JaTH160 is and crucial provide for the thecause possibility of high that mortality a combination mice. of amino acids of C 124 and NS3 482 of JaTH160 is crucial for the cause of high mortality in mice Simultaneous Substitutions of C124 and NS3482 are Not Enough to Cause High Mortality 3.3. Simultaneous We Substitutions predicted that of Csimultaneous 124 and NS3 482 substitutions are Not Enough of C124 to Cause and NS3482 High amino Mortality acids into S982-IC can increase mortality in mice. Therefore, to determine whether amino acids C124 and NS3482 We predicted that simultaneous substitutions of C of JaTH and NS3 482 amino acids into S982-IC can are responsible for the high mortality in mice, we inserted point mutations as containing C124 and increase mortality in mice. Therefore, to determine whether amino acids C 124 and NS3 482 of JaTH160 NS3482 of JaTH160 based on S982-IC (S-JC124NS3482) and examined the mortality in virus-infected mice are responsible for the high mortality in mice, we inserted point mutations as containing C 124 and (Figure 4C). However, this virus infection resulted in 20% mortality in mice (Figure 4C). NS3 482 of JaTH160 based on S982-IC (S-JC 124 NS3 482 ) and examined the mortality in virus-infected These results indicated that a combination of amino acids C124 and NS3482 of JaTH160 is not a mice (Figure 4C). However, this virus infection resulted in 20% mortality in mice (Figure 4C). critical factor of the higher mortality of JaTH160 compared with JaOArS982. These results indicated that a combination of amino acids C 124 and NS3 482 of JaTH160 is not a critical factor 4. Discussion of the higher mortality of JaTH160 compared with JaOArS Discussion In this study, to assess whether amino acid differences were crucial determinants for the different mortalities between JaTH160 and JaOArS982 infections in vivo, we attempted to specify the In this amino study, acids to assess responsible whether for amino the high acid mortality differences in mice were by crucial substituting determinants the amino for acids the different of JaOArS982 mortalities to between JaTH160. JaTH160 Based on and the stepwise JaOArS982 approach infections to narrow in vivo, down we attempted the genome toregion specify responsible the amino for the acids responsible high mortality for the of high JEV using mortality recombinant miceviruses by substituting derived from theinfectious amino acids cdna of clones, JaOArS982 we archived to JaTH160. Based two amino on the acid stepwise candidates approach of C124 to and narrow NS3482 down that likely the genome contribute region to the responsible increased mortality for the high in mice. mortality However, of JEV using simultaneous recombinant substitutions viruses derived of these from amino infectious acids based cdna on the clones, S982-IC wedid archived not result two in high amino acid mortality candidates like of JaTH-IC C 124 and virus NS3infection 482 that likely in mice. contribute From these to the observations, increased mortality we suggest in mice. that high However, mortality simultaneous due to substitutions JaTH160 virus of these infection amino cannot acids be based attributed on theto S982-IC a simple did mechanism not resultderived in highfrom mortality like specific JaTH-IC amino virus acids infection of JaTH160, in mice. and From that multiple these observations, and complex we mechanisms suggest that contribute high mortality to the high due to JaTH160 virus infection cannot be attributed to a simple mechanism derived from specific amino acids of JaTH160, and that multiple and complex mechanisms contribute to the high mortality

8 Viruses 2018, 10, 62 8 of 10 of JaTH160 virus infection in mice. We propose that a more sophisticated approach is required to elucidate the viral factors that determine the high virulence of JaTH160 in mice. S-JC 124 3a and S-J1aNS3 482 viruses induced high mortality in mice. However, the mechanism of the pathogenicity may be different between S-JC 124 3a and S-J1aNS3 482 viruses. In the case of the S-JC 124 3a virus, the combination of C 124 and the other amino acids or nucleotide sequences in the region of NS3 36 -NS4B 110 may be important for its pathogenicity. Moreover, the other amino acids located at close regions or separated regions could influence the C 124 -mediated viral pathogenicity. This theory is also applicable for S-J1aNS3 482 infection. It is predicted that such substitutions of amino acids and nucleotides may compensate for the function inducing the high mortality of each virus. However, it is likely to be too complex to determine such viral genome factors. A simple reductionist approach is unlikely to be the practical method of obtaining such data. We previously showed that JaTH160-infected mice developed severe infections of the CNS [9]. On the other hand, JaOArS982-infected mice exhibited varying degrees of encephalitis and different prognoses. We therefore proposed that fatal outcomes are attributable both to immunopathological changes and massive CNS infection [9]. However, it remains unclear whether severe CNS infection of JaTH160 is a direct cause of high mortality in mice or a result of a severe disease course including a specific immune response. In addition, our previous studies of JEV infection have shown that the mortality is not simply determined by neuroinvasiveness, because viral infections in the brains of surviving mice, that did not show apparent clinical signs, were similar levels of viral loads to those of dying mice [9]. These observations raise the possibility that different pathogenicity between JaOArS982 and JaTH160 infections may primarily be attributed to the peripherally induced host responses. Thus, both virus replication itself and host immune response induced by the virus infection should be focused on determining the viral factors for the fatal infection. We also previously showed the increase of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin (IL)-2, and IL-10 in the brain, and the decrease of TNF-α in the spleen in JaTH160 infected-mice [9]. From these observations we suggest that the varied disease symptoms in JE cases are primarily attributed to the immune response in individuals, and secondary viral factors contribute to the induction of such immune responses. Thus, to elucidate the mechanism of severe disease course based on the viral factors is still a priority in the development of effective treatment strategies for JE. Acknowledgments: This work was supported by JSPS KAKENHI Grant Numbers JP16H05174, JP , JP17H04661, JP15K15126, JP , and JP , and from the Japan Society for the Promotion of Science, Health and Labor Sciences Research Grant on Emerging and Re-emerging Infectious Diseases from the Japanese Ministry of Health, Labor and Welfare, Research on International Cooperation in Medical Science (Japan-US Cooperative Program), Health and Labor Sciences Research Grants, the Cooperative Research Grant(s) of NEKKEN, 2014, and the Japan Initiative for Global Research Network on Infectious Diseases. Author Contributions: Yuki Takamatsu, and Daisuke Hayasaka conceived and designed the experiments; Yuki Takamatsu, Leo Uchida., Muhareva Raekiansyah, and Daisuke Hayasaka performed the experiments; Yuki Takamatsu and Daisuke Hayasaka analyzed the data; Yuki Takamatsu, Mark Anthony Luz, Kouichi Morita and Daisuke Hayasaka wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. References 1. Ghosh, D.; Basu, A. Japanese encephalitis A pathological and clinical perspective. PLoS Negl. Trop. Dis. 2009, 3, e437. [CrossRef] 2. Solomon, T. Flavivirus encephalitis. N. Engl. J. Med. 2004, 351, [CrossRef] 3. Westaway, E.G.; Brinton, M.A.; Gaidamovich, S.; Horzinek, M.C.; Igarashi, A.; Kaariainen, L.; Lvov, D.K.; Porterfield, J.S.; Russell, P.K.; Trent, D.W. Flaviviridae. Intervirology 1985, 24, [CrossRef] 4. Gubler, D.J.; Kuno, G.; Markoff, L. Field's Virology, 5th ed.; Wolters Kluwer Lippincott Williams and Wilkins: Philadelphia, PA, USA, 2007; pp Sumiyoshi, H.; Hoke, C.H.; Trent, D.W. Infectious Japanese encephalitis virus RNA can be synthesized from in vitro-ligated cdna templates. J. Virol. 1992, 66,

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